A double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application thereof

Abstract

The application discloses a double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application thereof, and belongs to the technical field of biological technology, and aims to efficiently and sensitively detect double-stranded nucleic acid. The double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme comprises a double-stranded nucleic acid detection probe, lambda exonuclease and a reporter molecule, the double-stranded nucleic acid detection probe comprises one long single-stranded DNA named recognition chain R and n short single-stranded DNAs named Dz; the recognition chain R comprises a target binding domain and n Dz binding domains arranged in sequence from 5' to 3' in order; the 5' end of the recognition chain R is modified with a functional group; the Dz is a deoxyribozyme capable of cleaving the reporter molecule when the Dz is single-stranded, and the 5' end of the Dz is modified with sulfur. The application has high sensitivity and specificity, and the detection limit can be as low as a femtomole.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, specifically to a double-stranded DNA detection composition based on lambda exonuclease and deoxyribozyme and its application. BACKGROUND

[0002] In modern medicine, the application of nucleic acid detection technology is increasingly widespread, especially in the diagnosis and monitoring of infectious diseases. Direct detection of double-stranded nucleic acids is of great significance as it can provide more accurate pathogen identification and disease progression assessment. By enabling early diagnosis and personalized treatment, double-stranded nucleic acid detection not only reduces the rate of misdiagnosis, but also provides a reliable basis for clinical decision-making. The evolution of this technology has a profound impact on the field of public health, especially during outbreaks, rapid and accurate detection methods are crucial to enable timely public health measures.

[0003] In recent years, room temperature nucleic acid detection technology has gradually gained attention, its main advantage is that it does not require complex temperature control equipment, and can be performed at room temperature, thus greatly reducing the technical threshold and cost of detection. The room temperature nucleic acid detection method effectively simplifies the experimental process, making nucleic acid detection more feasible in resource-limited environments, especially in remote areas and developing countries. In particular, the application of the "one-pot approach" allows multiple experimental steps to be completed in the same reaction system, not only improving the convenience of operation, but also reducing the risk of sample handling and cross-contamination. By promoting this method, more extensive screening and monitoring can be achieved, thereby improving public health safety and enhancing the response capacity to potential epidemics.

[0004] Lambda exonuclease (λ exo) is an enzyme encoded by bacteriophage lambda, mainly involved in DNA repair and recombination processes. The enzyme has exonuclease activity, which can catalyze the step-by-step hydrolysis from the 5' end of double-stranded DNA, releasing 5'-mononucleotides. Due to its high efficiency and specificity, lambda exonuclease is widely used in molecular biology research and becomes a commonly used exonuclease. The specific probe modified by functional groups can directly target and recognize the target double-stranded target with the assistance of lambda exonuclease. When the probe binds to the target, it will trigger the release of functional single-stranded DNA, which can carry a fluorescent signal or realize signal amplification through the cutting characteristics of DNAzyme. DNAzyme is a kind of DNA molecule with catalytic function, which can catalyze various chemical reactions. Some DNAzymes can specifically cut RNA molecules as RNA-cleaving enzymes. By using the substrate containing specific RNA as the reporter molecule, the multi-round cutting characteristics of DNAzyme can realize signal amplification. This characteristic makes DNAzyme have important application potential in biological detection and molecular diagnosis, significantly improving the sensitivity and accuracy of detection. SUMMARY

[0005] The technical problem to be solved by the present application is how to efficiently and sensitively detect double-stranded nucleic acids.

[0006] To solve the above technical problems, the present application first provides a composition for double-stranded nucleic acid detection based on lambda exonuclease and DNAzyme, comprising a double-stranded nucleic acid detection probe, lambda exonuclease and a reporter molecule.

[0007] The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target is contained in the sample to be tested; the double-stranded nucleic acid detection probe comprises a single-stranded DNA named recognition strand R, and n single-stranded DNAs named Dz connected with the recognition strand R through base complementary pairing; the recognition strand R contains a target binding domain and n Dz binding domains arranged in order from 5' to 3' direction; n Dz is combined with the recognition strand R through n Dz binding domains to form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; the 5' end of the recognition strand R is modified with a functional group to realize the combination of the double-stranded nucleic acid detection probe and lambda exonuclease; the Dz is a DNAzyme that can cut the reporter molecule, and the 5' end of the Dz is modified to prevent the Dz from being degraded by lambda exonuclease;

[0008] The reporter molecule is a specific substrate of the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quenching group.

[0009] The fluorescence signal of the fluorescent group is quenched by the quenching group on the reporter molecule before the Dz enzyme is cleaved, and the fluorescence signal of the fluorescent group is not quenched by the quenching group after the Dz enzyme is cleaved.

[0010] The double-stranded nucleic acid detection probe is digested by λ exonuclease after recognizing the double-stranded target, and the Dz enzyme cleaves the reporter molecule, thereby releasing and amplifying the fluorescence signal.

[0011] In the above composition, the double-stranded target is a double-stranded DNA and / or a double-stranded DNA-RNA complex.

[0012] In the above composition, the Dz can be any deoxyribozyme capable of cleaving a specific substrate, which generally consists of a core catalytic domain and two substrate binding domains located on both sides of the core catalytic domain. The core catalytic domain has the activity of cleaving specific phosphodiester bonds, and the substrate binding domain is used for specific binding to the specific substrate. The phosphodiester bond is formed by ribonucleotides or deoxyribonucleotides.

[0013] In the above composition, the specific substrate is a DNA-RNA chimera or RNA.

[0014] In the above composition, the 5' end of the Dz is modified to be thio-modified, specifically, the 5' end is thio-modified at positions 3-5.

[0015] In the above composition, the deoxyribozyme can be selected from any one of 10-23 DNA enzyme, 8-17 DNA enzyme, 17E DNA enzyme (8-17 DNA enzyme variant), Mg5 DNA enzyme (8-17 DNA enzyme variant), 39E DNA enzyme, EtNa DNA enzyme, Ce13d DNA enzyme, etc.

[0016] The Dz can be any deoxyribozyme capable of cleaving a specific substrate, specifically, 10-23 DNA enzyme, 8-17 DNA enzyme, 17E DNA enzyme (8-17 DNA enzyme variant), Mg5 DNA enzyme (8-17 DNA enzyme variant), and other DNA enzymes, such as 39E DNA enzyme, EtNa DNA enzyme, Ce13d DNA enzyme, etc. (see Figure 1 (A) of WO 2018 / 018, 1 1 1 ).

[0017] The 10-23 DNAzyme is described in the non-patent literature "Lan T, Lu Y. Metal ion-dependent DNAzymes and their applications as biosensors. Interplay between Metal Ions and Nucleic Acids, 2011: 217-248."

[0018] The 10-23 DNAzyme is as shown in (A) of Figure 1 The core catalytic domain sequence is 5'-GGMTAGHNDNNNCGD-3', as shown in SEQ ID No: 1 of the sequence listing, positions 2-16, and has a length of 15 nucleotides, wherein the letter M represents A or C, H represents A, C or T, N represents A, C, G or T, and D represents A, G or T. The nucleotide closely connected to the 5' end of the core catalytic domain of the 10-23 DNAzyme is a purine deoxyribonucleotide, and R in the figure represents purine deoxyribonucleotide A or G. The 10-23 DNAzyme cleaves the phosphodiester bond between the pyrimidine ribonucleotide and the purine ribonucleotide, and Y in the figure represents pyrimidine ribonucleotide U or C, and R represents purine ribonucleotide A or G.

[0019] The 8-17 DNAzyme is described in the non-patent literature "Schlosser K, Li Y. A versatile endoribonuclease mimic made of DNA: characteristics and applications of the 8-17 RNA-cleaving DNAzyme. ChemBioChem, 2010, 11(7): 866-879."

[0020] The 8-17 DNAzyme is as shown in (A) of Figure 1As shown in (B), the core catalytic domain sequence of 8-17 DNAzyme is 5'-NNNNAGY(N)NNNNCGN(N)-3', with a length of 14-16 nucleotides, wherein (N) is one nucleotide N or no nucleotide, i.e., the core catalytic domain sequence of 8-17 DNAzyme is specifically any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', and 5'-NNNNAGYNNNNCGN-3', and the letter N represents A, C, G or T, and Y represents pyrimidine deoxyribonucleotide T or C. The 8-17 DNAzyme cleaves the phosphodiester bond between deoxyribonucleotides and ribonucleotides, and in the figure, N represents deoxyribonucleotides A, C, G or T, and rN represents ribonucleotides A, C, G or U.

[0021] The 17E DNAzyme (English name 17E DNAzyme) is a variant of 8-17 DNAzyme, which is described in the non-patent document "Zhou, W.; Zhang, Y.; Ding, J.; Liu, J., In vitro selection in serum: RNA-cleaving DNAzymes for measuring Ca 2+ and Mg 2+ . Acs Sensors 2016, 1 (5), 600-606.".

[0022] The Mg5 DNAzyme (English name Mg5 DNAzyme) is a variant of 8-17 DNAzyme, which is described in the non-patent document "Brown, A. K.; Li, J.; Pavot, C. M.-B.; Lu, Y., A lead-dependent DNAzyme with a two-step mechanism. Biochemistry 2003, 42 (23), 7152-7161.".

[0023] The 39E DNAzyme (English name 39E DNAzyme) is described in the non-patent document "Wu, P.; Hwang, K.; Lan, T.; Lu, Y., A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. Journal of the American Chemical Society 2013, 135 (14), 5254-5257."

[0024] The EtNa DNAzyme is described in the non-patent literature "Zhou, W.; Saran, R.; Chen, Q.; Ding, J.; Liu, J., A new Na + -dependent RNA-cleaving DNAzyme with over 1000-fold rate acceleration by ethanol. ChemBioChem 2016, 17 (2), 159-163.".

[0025] The Ce13d DNAzyme is described in the non-patent literature "Zhou, W.; Zhang, Y.; Huang, P.-J. J.; Ding, J.; Liu, J., A DNAzyme requiring two different metal ions at two distinct sites. Nucleic acids research 2016, 44(1), 354-363.".

[0026] In the composition, n is a natural number from 1 to 10 (n is 1, or n is a natural number from 2 to 10).

[0027] In the composition, the molar ratio of the recognition strand R and the Dz can be 1: n.

[0028] In the composition, the functional group can be any one of a phosphate group, a carboxyl group, an amino group, and a sulfhydryl group.

[0029] In the composition, the length of the thio modification is 3-5 nucleotides.

[0030] In the composition, the quencher group can be BHQ1; and the fluorescent group can be FAM.

[0031] In the composition, the specific composition can be any one of P1-P7:

[0032] P1, the double strand consisting of the single stranded DNA with sequence of SEQ ID No: 8 and the single stranded DNA with sequence of SEQ ID No: 9 or the single stranded RNA with sequence of SEQ ID No: 10 is the double stranded target, the double stranded nucleic acid detection probe is combined by one single stranded DNA with sequence of SEQ ID No: 2 and one single stranded DNA with sequence of SEQ ID No: 4 through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with sequence of SEQ ID No: 6;

[0033] P2, the double strand consisting of the single stranded DNA with sequence of SEQ ID No: 8 and the single stranded DNA with sequence of SEQ ID No: 9 or the single stranded RNA with sequence of SEQ ID No: 10 is the double stranded target, the double stranded nucleic acid detection probe is combined by one single stranded DNA with sequence of SEQ ID No: 3 and one single stranded DNA with sequence of SEQ ID No: 5 through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with sequence of SEQ ID No: 7;

[0034] P3, the double strand consisting of the single stranded DNA with sequence of SEQ ID No: 8 and the single stranded DNA with sequence of SEQ ID No: 9 or the single stranded RNA with sequence of SEQ ID No: 10 is the double stranded target, the double stranded nucleic acid detection probe is combined by one single stranded DNA with sequence of SEQ ID No: 11 and two single stranded DNAs with sequence of SEQ ID No: 4 through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with sequence of SEQ ID No: 6;

[0035] P4, the double strand consisting of the single stranded DNA with sequence of SEQ ID No: 8 and the single stranded DNA with sequence of SEQ ID No: 9 or the single stranded RNA with sequence of SEQ ID No: 10 is the double stranded target, the double stranded nucleic acid detection probe is combined by one single stranded DNA with sequence of SEQ ID No: 12 and one single stranded DNA with sequence of SEQ ID No: 4 through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with sequence of SEQ ID No: 6;

[0036] P5, the double strand consisting of the single stranded DNA with the sequence of SEQ ID No: 8 and the single stranded DNA with the sequence of SEQ ID No: 9 or the single stranded RNA with the sequence of SEQ ID No: 10 is the double stranded target, the double stranded nucleic acid detection probe is formed by the single stranded DNA with the sequence of SEQ ID No: 13 and the single stranded DNA with the sequence of SEQ ID No: 4 combined through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No: 6;

[0037] P6, the double strand consisting of the single stranded DNA with the sequence of SEQ ID No: 8 and the single stranded DNA with the sequence of SEQ ID No: 9 is the double stranded target, the double stranded nucleic acid detection probe is formed by the single stranded DNA with the sequence of SEQ ID No: 14 and the single stranded DNA with the sequence of SEQ ID No: 4 combined through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No: 6;

[0038] P7, the double strand consisting of the single stranded DNA with the sequence of SEQ ID No: 15 1-45 and the single stranded DNA with the sequence of the reverse complement of SEQ ID No: 15 1-45 is the double stranded target, the double stranded nucleic acid detection probe is formed by the single stranded DNA with the sequence of SEQ ID No: 15 and the single stranded DNA with the sequence of SEQ ID No: 4 combined through base complementary pairing, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No: 6.

[0039] The application also provides the above double stranded nucleic acid detection probe.

[0040] The application also provides a method for detecting whether a target double stranded nucleic acid is contained in a sample to be detected, which comprises reacting the double stranded nucleic acid detection probe with the sample to be detected in the presence of lambda exonuclease and the reporter molecule, detecting the fluorescence signal of the reaction system, and determining whether the target double stranded nucleic acid is contained in the sample to be detected according to the fluorescence signal.

[0041] In the above method, the determination of whether the target double stranded nucleic acid is contained in the sample to be detected according to the fluorescence signal can be specifically that if the fluorescence signal changes (significantly changes), the sample to be detected contains the target double stranded nucleic acid; and if the fluorescence signal does not change (significantly changes), the sample to be detected does not contain the target double stranded nucleic acid.

[0042] The application also provides a product consisting of X1 and X2; the X1 is the composition or the double stranded nucleic acid detection probe, and the X2 is a reagent and / or instrument required for detection.

[0043] In the product, the instrument can be a real-time fluorescence detector.

[0044] In the product, the reagent required for the detection also needs to contain metal ions that make the Dz have deoxyribozyme activity as a cofactor. 2+ , Mn 2+ , Ca 2+ , Ba 2+ and Sr 2+ , etc.; the cofactor of 8-17 DNAzyme can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ , etc.; the cofactor of 17E DNAzyme (8-17 DNAzyme variant) can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ , etc.; the cofactor of Mg5 DNAzyme (8-17 DNAzyme variant) can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ , etc.; the cofactor of 39E DNAzyme can be UO 2+ ; the cofactor of EtNa DNAzyme can be selected from Na + , Ca 2+ ; the cofactor of Ce13d DNAzyme can be Na + .

[0045] To solve the above technical problems, the application also provides applications of the composition, the double-stranded nucleic acid detection probe, the method and the product in the preparation of nucleic acid detection reagents.

[0046] The application discloses a double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application thereof. The double-stranded nucleic acid detection composition comprises a double-stranded nucleic acid detection probe, the main body of the double-stranded nucleic acid detection probe is a single-stranded DNA with a 5' end modified by a phosphate group, a carboxyl group and an amino group and the like, and the double-stranded nucleic acid detection probe can specifically recognize and target a target double-stranded nucleic acid (i.e. a double-stranded target such as double-stranded DNA, a double-stranded DNA-RNA complex) with the assistance of lambda exonuclease (lambda exonuclease). After the probe is combined with the target, the release of a functional single-stranded DNA is triggered, the functional single-stranded DNA is a deoxyribozyme, and the deoxyribozyme can cut a specific substrate with quenched fluorescent groups multiple times, so that the release and amplification of a fluorescent signal are realized, and the intensity of the fluorescent signal is significantly enhanced. A remarkable feature of the detection method is that the detection of dsDNA in clinical samples (such as cfDNA in blood, pathogen DNA, DNA in tissue samples and body fluid samples and the like) can be realized without pre-amplification of the to-be-detected substance, and the detection limit can be as low as femtomolar. The application has high sensitivity and specificity, is widely applicable to molecular biology, clinical diagnosis and other related fields, and has important scientific research and clinical application values. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 A schematic diagram for specific detection of double-stranded nucleic acid and signal amplification of the type I detection probe constructed in Example 1 and a fluorescence-time trajectory diagram are shown in (A) of the figure. Figure 1 (A) of the figure is a schematic diagram of the 10-23 DNA enzyme cited in Example 1, the upper strand is a specific substrate, and the lower strand is the 10-23 DNA enzyme, the 10-23 DNA enzyme comprises a nucleotide sequence of 5'-RGGMTAGHNDNNNCGD-3' (see SEQ ID No: 1), wherein the letter R represents a purine deoxyribonucleotide A or G, the letter M represents A or C, H represents A, C or T, N represents A, C, G or T, and D represents A, G or T; the core catalytic domain sequence of the 10-23 DNA enzyme is 5'-GGMTAGHNDNNNCGD-3', as shown in SEQ ID No: 1 in the sequence listing, and the length is 15 nucleotides; the 10-23 DNA enzyme cuts the phosphodiester bond between a pyrimidine ribonucleotide and a purine ribonucleotide, and in the figure, the letter Y represents a pyrimidine ribonucleotide U or C, and the letter R represents a purine ribonucleotide A or G. Figure 1(B) is a schematic diagram of the 8-17 DNA enzyme cited in Example 1, the upper strand is a specific substrate, the lower strand is the 8-17 DNA enzyme, the core catalytic domain sequence of the 8-17 DNA enzyme is 5'-NNNNAGY(N)NNNNCGN(N)-3', the length is 14-16 nucleotides, wherein (N) is one nucleotide N or no nucleotide, i.e. the core catalytic domain sequence of the 8-17 DNA enzyme is any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', 5'-NNNNAGYNNNNCGN-3', the letter N represents A, C, G or T, and Y represents a pyrimidine deoxyribonucleotide T or C; the 8-17 DNA enzyme cleaves the phosphodiester bond between deoxyribonucleotides and ribonucleotides, and in the figure, N represents deoxyribonucleotides A, C, G or T, and rN represents ribonucleotides A, C, G or U. Figure 1 (C) is a schematic diagram of the detection of double-stranded DNA by the type I detection probe formed by the combination of the recognition chain R and Dz at a ratio of 1:1, and the release of Dz equivalent to R; Figure 1 (D) and Figure 1 (F) are respectively a schematic diagram of the cleavage cycle of the reporter molecule S1 by Dz1 (10-23 DNA enzyme) released by the reaction of the probe P1 and the double-stranded target, and a fluorescence-time trajectory diagram generated for the DNA double-stranded target and the DNA-RNA complex target respectively; Figure 1 (E) and Figure 1 (G) are respectively a schematic diagram of the cleavage cycle of the reporter molecule S2 by Dz2 (8-17 DNA enzyme) released by the reaction of the probe P2 and the double-stranded target, and a fluorescence-time trajectory diagram generated for the DNA double-stranded target and the DNA-RNA complex target respectively.

[0048] Figure 2 is a schematic diagram of the specific detection of double-stranded DNA by the type II detection probe constructed in Example 2 and the amplification of the signal, and a fluorescence-time trajectory diagram. Among them, Figure 2 (A) is a schematic diagram of the cleavage cycle of the reporter molecule S1 by Dz1 (10-23 DNA enzyme) released by the reaction of the type II detection probe P3 formed by the combination of the recognition chain R and Dz at a ratio of 1:n (n = 1-10, n = 2 in Example 3) and the double-stranded DNA, wherein n (n = 2 in Example 3) times the amount of Dz1 (10-23 DNA enzyme) is equivalent to the amount of R; Figure 2 (B) is a fluorescence-time trajectory diagram and a columnar diagram generated by the detection of double-stranded DNA by the type II detection probe P3.

[0049] Figure 3Schematic diagram of detection using probe P for detecting double-stranded nucleic acid and fluorescence-time trajectory graph for Example 3. Wherein, Figure 3 (A) of the above is a modification of the 5' end of the recognition strand R of the probe for detecting double-stranded nucleic acid using different functional groups; Figure 3 (B) of the above is a schematic diagram of detection using probe P with recognition strand R modified with different groups; Figure 3 (C) of the above is a fluorescence-time trajectory graph generated by detection using probe P with recognition strand R modified with different groups.

[0050] Figure 4 Fluorescence-time trajectory graph and columnar graph for detecting non-small cell lung cancer related genes using double-stranded nucleic acid detection probe P1 of Type I in Example 4. DETAILED DESCRIPTION

[0051] The present application will be further described in conjunction with the specific embodiments below, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0052] In the quantitative test in the following examples, three repeated experiments were set up, and the average value was taken.

[0053] In the experimental methods in the following examples, unless otherwise specified, they are all conventional methods. In the following examples, unless otherwise specified, the materials, reagents, etc. used can be obtained from commercial channels.

[0054] The nucleic acid sequences in the following examples were synthesized by Shanghai Shengong Bioengineering (Shanghai) Co., Ltd.

[0055] The 1 × lambda exonuclease buffer in the following examples is a product of New England Biolabs (NEB) Co., Ltd., with product number M0262S. The reagent contains exonuclease (lambda exonuclease).

[0056] Example 1

[0057] 1. Design of Type I double-stranded nucleic acid detection probe and signal amplification system

[0058] The Type I double-stranded nucleic acid detection probe and signal amplification system provided in the present embodiment comprises a double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme.

[0059] The composition comprises a double-stranded nucleic acid detection probe, lambda exonuclease and a reporter molecule;

[0060] The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target is contained in the sample to be detected; the double-stranded nucleic acid detection probe comprises a single-stranded DNA named recognition strand R and n single-stranded DNAs named Dz connected with the recognition strand R through base complementary pairing; the recognition strand R contains a target binding domain and n Dz binding domains arranged in sequence from 5' to 3' direction; n Dz binding domains and the recognition strand R form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; the 5' end of the recognition strand R is modified with a functional group to realize the binding of the double-stranded nucleic acid detection probe and lambda exonuclease; the Dz is a deoxyribozyme capable of enzymatically cutting the reporter molecule, and the 5' end of the Dz is modified to prevent the Dz from being degraded.

[0061] The reporter molecule is a specific substrate of the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quenching group. The quenching group on the reporter molecule quenches the fluorescent signal of the fluorescent group before being enzymatically cut by the Dz, and the quenching group does not quench the fluorescent signal of the fluorescent group after being enzymatically cut by the Dz.

[0062] In this embodiment, n = 1, and the molar ratio of the recognition strand R to the Dz is 1:1. Hereinafter, the double-stranded nucleic acid detection probe with the molar ratio of the recognition strand R to the Dz being 1:1 is referred to as a type I double-stranded nucleic acid detection probe.

[0063] The sulfur modification can be modification of 3-5 nucleotides, and in this embodiment, 3 nucleotides are modified.

[0064] The modification group can be any one of a phosphate group, a carboxyl group, an amino group, and a sulfhydryl group. In this embodiment, carboxylation modification is used.

[0065] The Dz can be specifically selected from any one of 10-23 DNA enzyme, 8-17 DNA enzyme, 17E DNA enzyme (8-17 DNA enzyme variant), Mg5 DNA enzyme (8-17 DNA enzyme variant), 39E DNA enzyme, EtNa DNA enzyme, Ce13d DNA enzyme, etc. In this embodiment, two type I detection probes named P1 and P2 are designed, wherein P1 uses 10-23 DNA enzyme, and P2 uses 8-17 DNA enzyme:

[0066] The detection probe P1 is used to detect the DNA double-stranded target composed of T1 and T2 in Table 1, or to detect the DNA-RNA complex target composed of T1 and T3 in Table 1. It is composed of a recognition strand R1 and a short single-stranded Dz1, which can enzymatically cut a specific substrate S1. The referenced DNA enzyme is, for example, Figure 1The 10-23 DNase shown in (A) is an example. After the detection probe P1 recognizes the double-stranded target, the recognition strand R1 is digested by the λ exonuclease (λ exonuclease) to release an equal amount of 10-23 DNase (as a short single-stranded Dz1) to the recognition strand R1. The 10-23 DNase enters the cycle to cleave the specific substrate S1, which serves as a reporter molecule, to generate and amplify the signal.

[0067] The sequence of R1 is shown in SEQ ID No:2 in the sequence listing. It is 72 nucleotides in length and has a carboxylic acid group modified at position 1. R1 is formed by connecting a target binding domain (positions 1-45) and a Dz binding domain (positions 46-72) along the 5'-3' direction. The target binding domain is anticomplementary to positions 21-65 of one strand T1 of the double-stranded target, and the Dz binding domain is anticomplementary to Dz1.

[0068] Dz1 uses 10⁻²³ DNA enzyme, and its schematic diagram is as follows: Figure 1 As shown in (A), the 10-23 DNase consists of a core catalytic domain and two substrate-binding domains located on either side of the core catalytic domain. The sequence of the core catalytic domain of the 10-23 DNase is 5'-GGMTAGHNDNNNCGD-3', as shown in positions 2-16 of SEQ ID No:1 in the sequence listing, and is 15 nucleotides in length, where M represents A or C, H represents A, C, or T, N represents A, C, G, or T, and D represents A, G, or T. The nucleotide tightly connected at the 5' end of the core catalytic domain of the 10-23 DNase is a purine deoxyribonucleotide, and R in the figure represents purine deoxyribonucleotide A or G. The 10-23 DNase cleaves the phosphodiester bond between pyrimidine ribonucleotides and purine ribonucleotides, and Y in the figure represents pyrimidine ribonucleotide U or C, and R represents purine ribonucleotide A or G. In this embodiment, the core catalytic domain sequence of the 10-23 DNase is GGCTAGCTACAACGA (as shown in positions 6-21 of SEQ ID No:4). Each flanking the catalytic domain is a 6 nt substrate-binding domain for the recognition and binding of the specific substrate S1. The 5' end of Dz1 is thiolated. The specific sequence of the 10-23 DNase used in this embodiment is shown in SEQ ID No:4 in the sequence listing, with a length of 27 nucleotides. Positions 46-72 of the 10-23 DNase are anticomplementary to positions 46-72 of R1; positions 1-6 of the 10-23 DNase are anticomplementary to positions 8-13 of S1, and positions 22-27 are anticomplementary to positions 1-6 of S1; positions 1-3 of the 10-23 DNase are thiolated.

[0069] The 10-23 DNA enzyme can cleave the specific substrate S1, S1 is a DNA-RNA chimera with sequence as shown in SEQ ID No: 6 in the sequence listing. S1 is 13 nucleotides long, containing a Dz1 cleavage recognition sequence purine ribonucleotide and pyrimidine ribonucleotide rGrU, the 5' end T is modified by FAM, and the 3' end A is modified by BHQ1.

[0070] Detection probe P2: for detecting the DNA double-stranded target consisting of T1 and T2 in Table 1, or for detecting the DNA-RNA complex target consisting of T1 and T3 in Table 1. Consists of recognition strand R2 and short single-stranded Dz2, Dz2 can specifically cleave the specific substrate S2, the referenced DNA enzyme is 8-17 DNA enzyme as shown in (B) of Figure 1 After the detection probe P2 recognizes the double-stranded target, the recognition strand R2 is digested by λ exonuclease (λ exonuclease) and releases an equal amount of 8-17 DNA enzyme (as short single-stranded Dz2) to the recognition strand R2, which enters the cycle of cleaving S2 as a reporter molecule to generate and amplify the signal.

[0071] The sequence of R2 is shown in SEQ ID No: 3 in the sequence listing, which is 78 nucleotides long, and the first position is modified with a carboxyl group. R2 is connected in the 5'-3' direction by a target binding domain (1-45) and a Dz binding domain (46-78); wherein the target binding domain is reverse complementary to the 21-65 of one strand T1 of the double-stranded target, and the Dz binding domain is reverse complementary to the Dz2.

[0072] Dz2 adopts 8-17 DNA enzyme, the schematic diagram is as shown in Figure 1The 8-17 DNA enzyme consists of a core catalytic domain and two substrate binding domains flanking the core catalytic domain. The sequence of the core catalytic domain of the 8-17 DNA enzyme is 5'-NNNNAGY(N)NNNNCGN(N)-3', with a length of 14-16 nucleotides, wherein (N) is one nucleotide N or no nucleotide, i.e. the sequence of the core catalytic domain of the 8-17 DNA enzyme is specifically any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', 5'-NNNNAGYNNNNCGN-3', and the letter N represents A, C, G or T, and Y represents pyrimidine deoxyribonucleotide T or C. The 8-17 DNA enzyme cleaves the phosphodiester bond between a deoxyribonucleotide and a ribonucleotide, and in the figure, N represents deoxyribonucleotide A, C, G or T, and rN represents ribonucleotide A, C, G or U. The sequence of the core catalytic domain of the 8-17 DNA enzyme used in this example is TCCGAGCCGGTCGAA (as shown in SEQ ID No: 5, positions 10-24), and each of the two substrate binding domains flanking the catalytic domain has a length of 9 nt, for the recognition and binding of the specific substrate S2; and the 5' end of the Dz2 is thio-modified. The specific sequence of the 8-17 DNA enzyme used in this example is shown in SEQ ID No: 5 in the sequence listing, with a length of 33 nucleotides, and the 8-17 DNA enzyme is reverse complementary to positions 46-78 of R2; the 8-17 DNA enzyme is reverse complementary to positions 12-20 of S2 at positions 1-9, and reverse complementary to positions 1-9 of S2 at positions 25-33; and the 8-17 DNA enzyme is thio-modified at positions 1-3.

[0073] The 8-17 DNA enzyme can cleave the specific substrate S2, and S2 is a DNA-RNA chimera with a sequence as shown in SEQ ID No: 7 in the sequence listing. S2 has a length of 20 nucleotides, and contains a cleavage recognition sequence purine ribonucleotide and pyrimidine deoxyribonucleotide rAdG of Dz2 (the catalytic center is TCCGAGCCGGTCGAA), the 5' terminal A is modified by FAM, and the 3' terminal G is modified by BHQ1.

[0074]

[0075]

[0076] Note: in Table 1, * indicates a phosphorothioate backbone linkage, specifically, a non-bridging oxygen atom on the phosphodiester bond between the two adjacent nucleotides is replaced by a sulfur atom to form a phosphorothioate group, and the structure is formula (1); the nucleotides connected by the phosphorothioate group are shown in formula (2):

[0077] Formula (1);

[0078] Formula (2).

[0079] Figure 1 Figure (C) of the present application is a schematic diagram of the reaction of the I-type detection probe recognizing and processing double-stranded target (DNA double-stranded target and DNA-RNA complex target): the target binding domain of the recognition strand R recognizes T1 in the double-stranded target and binds with it to form double-stranded DNA with a 5'-COOH end, which is subjected to enzymatic digestion reaction under the action of lambda exonuclease, and the double-stranded DNA with a 5'-COOH end is digested to release Dz.

[0080] The released Dz (D) in Figure (D) of the present application is represented by 10-23 DNAzyme, Figure 1 The 10-23 DNAzyme in Figure (D) of the present application, Figure 1 The 8-17 DNAzyme in Figure (E) of the present application) has deoxyribonuclease activity in the presence of magnesium ions, can enzymatically cut the substrate (specific substrate S, the substrate of 10-23 DNAzyme is S1, and the substrate of 8-17 DNAzyme is S2), produce a fluorescence signal, and circulate to realize amplification of the fluorescence signal.

[0081] 2. Preparation of the I-type double-stranded nucleic acid detection probe and signal amplification system

[0082] 2.1 Preparation of the I-type detection probe

[0083] Preparation of the I-type detection probe P1:

[0084] According to Table 1, single-stranded DNA is artificially synthesized and modified to obtain R1 and Dz1.

[0085] The reaction buffer environment is 1 × lambda exonuclease buffer, and under normal temperature conditions, R1 and Dz1 are added to synthesize the I-type detection probe P1, and the rest of the volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of R1 is 1 μM, and the concentration of Dz1 is 1 μM. The annealing program is used to ensure the correct combination of R1 and Dz1 to obtain the I-type double-stranded nucleic acid detection probe P1.

[0086] The annealing program setting program is as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0087] Preparation of the I-type detection probe P2:

[0088] According to Table 1, single-stranded DNA is artificially synthesized and modified to obtain R2 and Dz2.

[0089] The reaction buffer environment is 1 × lambda exonuclease buffer, and under normal temperature conditions, R2 and Dz2 are added to synthesize type I detection probe P2, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of R1 is 1 μM, and the concentration of Dz2 is 1 μM. The annealing program is used to ensure that R2 and Dz2 are correctly combined to obtain type I double-stranded nucleic acid detection probe P2.

[0090] The annealing program setting program is as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0091] 2.2 Preparation of double-stranded target

[0092] The double-stranded target used is a DNA double-stranded target composed of T1 and T2, or a DNA-RNA complex target composed of T1 and T3. The preparation method is as follows:

[0093] The single-stranded nucleic acid is artificially synthesized and modified according to Table 1 to obtain T1, T2, and T3.

[0094] The reaction buffer environment is 1 × lambda exonuclease buffer, and under normal temperature conditions, T1 and T2 are added to synthesize DNA double-stranded target, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of T1 in the system is 500 nM, and the concentration of T2 is 500 nM. The annealing program is used to ensure that T1 and T2 are correctly combined to obtain DNA double-stranded target.

[0095] The reaction buffer environment is 1 × lambda exonuclease buffer, and under normal temperature conditions, T1 and T3 are added to synthesize DNA-RNA complex target, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of T1 in the system is 500 nM, and the concentration of T2 (T3) is 500 nM. The annealing program is used to ensure that T1 and T3 are correctly combined to obtain DNA-RNA complex target.

[0096] The annealing program setting program is as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0097] 2.3 Establishment of type I double-stranded nucleic acid detection probe and signal amplification system

[0098] The DNA double-stranded target system and the DNA-RNA complex target system prepared in 2.2 above are respectively taken as the to-be-tested targets of the present example. The double-stranded target prepared in 2.2 above is diluted into a concentration gradient (the specific concentrations are 5 pM, 10 pM, 15 pM, 50 pM, and 100 pM) as the to-be-tested sample, and a blank control is set.

[0099] 2 μL of the type I detection probe P1 with a concentration of 1 μM is taken, so that the concentration in the detection system is 100 nM. 2 μL of the to-be-tested sample is added, and λ exonuclease is added so that the concentration in the system is 125 U / mL. The artificially synthesized specific substrate S1 is added so that the concentration in the detection system is 500 nM. If the total volume of the system is less than 20 μL, ddH2O is supplemented. It is placed in a real-time fluorescence detector under the condition of 37 °C for detection. The detection time interval is set to 5 seconds for one cycle, the cycle number is 1440, and the detection time length is 120 minutes. The fluorescence intensity-time trajectory is shown in (F) of FIG. 4. Figure 1

[0100] 2 μL of the type I detection probe P2 with a concentration of 1 μM is taken, so that the concentration in the detection system is 100 nM. 2 μL of the to-be-tested sample is added, and λ exonuclease is added so that the concentration in the system is 125 U / mL. The artificially synthesized specific substrate S2 is added so that the concentration in the detection system is 500 nM. If the total volume of the system is less than 20 μL, ddH2O is supplemented. It is placed in a real-time fluorescence detector under the condition of 37 °C for detection. The detection time interval is set to 10 seconds for one cycle, the cycle number is 720, and the detection time length is 120 minutes. The fluorescence intensity-time trajectory is shown in (G) of FIG. 5. Figure 1

[0101] The fluorescence value-time trajectory of the reaction process is captured and recorded by a real-time fluorescence instrument, and is output as a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0102] Figure 1 The experimental results in (F) and (G) of FIGS. 4 and 5 show that when there is no double-stranded target in the to-be-tested sample, the fluorescence value remains at a low level and no obvious change is observed. When there is a double-stranded target in the sample, the fluorescence value increases significantly with time. The higher the concentration of the double-stranded target in the to-be-tested sample, the greater the fluorescence value, showing a positive correlation between the concentration and the fluorescence signal. This shows that the detection method has good sensitivity in recognizing and quantifying double-stranded targets.

[0103] Example 2

[0104] 1. Design of type II double-stranded nucleic acid detection probe and signal amplification system ​​

[0105] In this embodiment, a type II double-stranded nucleic acid detection probe is designed based on the type I detection probe in Embodiment 1.

[0106] The type II double-stranded nucleic acid detection probe is composed of one recognition strand R and n short single-stranded Dz, and n is a natural number from 2 to 10. The molar ratio of the recognition strand R to Dz in the type II detection probe is 1: n, and n is 2 in this embodiment.

[0107] The modification group is any one of a phosphate group, a carboxyl group and an amino group. Carboxylation modification is used in this embodiment.

[0108] After the type II double-stranded DNA probe in this embodiment recognizes the double-stranded target, the recognition strand R is digested by λ exonuclease and releases twice the amount of Dz, which enters the cycle of the enzyme-specific substrate S to generate and amplify the fluorescence signal.

[0109] In this embodiment, a type II double-stranded nucleic acid detection probe named P3 is specifically designed:

[0110] The detection probe P3 is used for detecting the double-stranded target composed of T1 and T2 in Table 1, which is composed of one recognition strand R3 (see Table 2) and two short single-stranded Dz1 (see Table 1). The short single-stranded Dz1 can be used to digest the enzyme-specific substrate S1 (see Table 1), and the DNA enzyme used is 10-23 DNA enzyme as shown in (B) of Figure 1 After the detection probe P3 recognizes the double-stranded target, the recognition strand R3 is digested by λ exonuclease and releases twice the amount of Dz1 than the recognition strand R2, which enters the cycle of the enzyme-specific substrate S to generate and amplify the fluorescence signal.

[0111] The sequence of R3 is shown in SEQ ID No: 11 in the sequence listing, and the length is 99 nucleotides. The first position is modified with a carboxylic acid group. R3 is connected in the 5'-3' direction by a target binding domain (1-45), two consecutive Dz binding domains (46-72, 73-99). The target binding domain is reverse complementary to the 21-65 of one strand T1 in the double-stranded target, and the two Dz binding domains are both reverse complementary to the Dz1.

[0112] The sequences of the recognition strand R3, Dz1, reporter molecule S1 and double-stranded target-T (composed of T1 and T2) involved in this embodiment are shown in Table 2.

[0113]

[0114] Note: COOH in Table 2 represents carboxylic acid modification.

[0115] Figure 2The reaction schematic diagram of the (A) of the II type detection probe recognizing the double-stranded nucleic acid is as follows: the target binding domain of the recognition chain R recognizes T1 in the double-stranded target and combines with it to form double-stranded DNA with a 5'-COOH end, and the enzyme digestion reaction occurs under the action of lambda exonuclease, the double-stranded DNA with a 5'-COOH end is digested, and 2 times of Dz (10-23 DNA enzyme in this embodiment) of the recognition chain R is released.

[0116] The released Dz (10-23 DNA enzyme in this embodiment) has deoxyribonuclease activity in the presence of magnesium ions, can perform enzyme cutting on the substrate (specific substrate S, the substrate of 10-23 DNA enzyme is S1), produce a fluorescence signal, and perform a cycle to realize amplification of the fluorescence signal.

[0117] 2. Preparation of the II type double-stranded nucleic acid detection probe and the signal amplification system

[0118] 2.1 Preparation of the II type double-stranded nucleic acid detection probe

[0119] According to Table 2, the single-stranded DNA is artificially synthesized and modified to obtain R3, and according to Table 1, the single-stranded Dz1 is artificially synthesized and modified.

[0120] The reaction buffer environment is 1 × lambda exonuclease buffer, under normal temperature conditions, R3 and Dz1 are added to synthesize the I type detection probe, and the rest of the volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of R2 is 1 μM, and the concentration of Dz is 2 μM. The annealing program is used to ensure that R3 and Dz1 are correctly combined to obtain the II type double-stranded nucleic acid detection probe P3.

[0121] The annealing program setting program is as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0122] 2.2 Preparation of the double-stranded target

[0123] The double-stranded target used is a DNA double-stranded target composed of T1 and T2. The preparation method is shown in Example 1, 2.2.

[0124] 2.3 Establishment of the II type double-stranded nucleic acid detection probe and the signal amplification system

[0125] The double-stranded target prepared in 2.2 above is used as the target to be detected in this example. The double-stranded target prepared in 2.2 above is diluted to a concentration gradient (specific concentrations are 500 fM, 1 pM, 10 pM, 50 pM, 100 pM, 200 pM, 500 pM, and 1 nM) as the sample to be detected, and a blank control is set.

[0126] 2 μL of the type II double-stranded nucleic acid detection probe P3 with a concentration of 1 μM is taken, and the concentration in the detection system is 100 nM. 2 μL of the sample to be detected is added, and λ exonuclease is added to make the concentration in the system 125 U / mL. The synthetic specific substrate S1 is added to make the concentration in the detection system 500 nM. If the total volume of the system is less than 20 μL, ddH2O is supplemented. The system is placed in a real-time fluorescence detector at 37°C for detection. The detection time interval is set to 10 seconds per cycle, the cycle number is 1440, and the detection time is 240 minutes. The fluorescence intensity-time trajectory is obtained.

[0127] The fluorescence value-time trajectory of the reaction process is captured and recorded by a real-time fluorescence instrument, and the output is a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0128] Figure 2 The experimental results of the middle (B) show that when there is no double-stranded target in the sample to be detected, the fluorescence value remains stable and does not change significantly. When there is a double-stranded target in the sample, the fluorescence value increases significantly with time, indicating the activity of the reaction. In addition, the higher the concentration of the double-stranded target in the sample to be detected, the higher the fluorescence value, further proving the positive correlation between the concentration and the fluorescence signal. This indicates that the detection technology has good sensitivity and specificity in recognizing and quantifying double-stranded targets.

[0129] Example 3

[0130] 1. Design of type I double-stranded nucleic acid detection probes modified by different groups

[0131] In this example, type I double-stranded nucleic acid detection probes modified by different groups are designed based on the type I detection probe P1 in Example 1. In this example, four type I detection probes named P1, P4, P5, and P6 are specifically designed according to the recognition strands R of different modification groups:

[0132] The detection probe P1 is the detection probe P1 in the above-mentioned Example 1.

[0133] The composition of detection probes P4, P5, and P6 is the same as that of detection probe P1, the difference being the modification groups of the recognition chain R. The 5' end of R1 is modified with carboxylation, the 5' end of R4 is modified with carboxyphosphorylation, the 5' end of R5 is modified with aminoation, and the 5' end of R6 is modified with thiolation.

[0134]

[0135] Note: In Table 3, COOH indicates carboxylation modification (5' carboxyl modification); P indicates phosphorylation modification (5' phosphorylation modification); NH2 indicates amination modification (5' amino modification); and HS indicates thiolation modification (5' thiolation modification).

[0136] Figure 3 (A) is a schematic diagram of the reaction in which the detection probe P recognizes and processes the double-stranded DNA target:

[0137] like Figure 3 As shown in (B), the R ends of the recognition strands of the three probes are modified with carboxylic acid groups, phosphate groups, amino groups, and thiol groups, respectively. In the presence of λ exonuclease, the target binding domain of the recognition strand R recognizes T1 in the double-stranded target and binds to it to form a double-stranded DNA with functional groups modified at the 5' end. Under the action of λ exonuclease, an enzymatic digestion reaction occurs, and the double-stranded DNA with functional groups modified at the 5' end is digested, releasing Dz.

[0138] The released Dz1 has DNase activity in the presence of magnesium ions, can enzymatically cleave the substrate S1, generate a fluorescent signal, and amplify the fluorescent signal through cycling.

[0139] 2. Preparation of Type I double-stranded nucleic acid detection probe and signal amplification system

[0140] 2.1 Preparation of Type I Detection Probe

[0141] Preparation of type I detection probes P (P1, P4, P5, P6):

[0142] Single-stranded DNA was artificially synthesized and modified according to Table 3 to obtain R (R1, R4, R5, R6), and single-stranded Dz1 was artificially synthesized and modified according to Table 1.

[0143] The reaction buffer environment is 1 x lambda exonuclease buffer. At room temperature, R (R1, R4, R5, R6) and Dz1 are added to synthesize type I detection probe P (P1, P4, P5, P6). The rest of the volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of R (R1, R4, R5, R6) is 1 μM, and the concentration of Dz1 is 1 μM. An annealing program is used to ensure that R (R1, R4, R5, R6) and Dz1 are correctly combined to obtain type I double-stranded nucleic acid detection probe P1.

[0144] The annealing program setting program is as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0145] 2.2 Preparation of double-stranded target

[0146] The double-stranded target used is a DNA double-stranded target composed of T1 and T2. The preparation method is shown in Example 1, 2.2.

[0147] 2.3 Establishment of type I double-stranded nucleic acid detection probe and signal amplification system

[0148] The double-stranded target prepared in the above 2.2 is used as the target to be detected in this example. The double-stranded target prepared in the above 2.2 is diluted to 5 nM as the sample to be detected, and a blank control is set.

[0149] The detection system of the four probes modified by different functional groups is constructed as follows: 1 μL of detection probe (P1, P4, P5 or P6) with a concentration of 1 μM is taken to make the concentration in the detection system 50 nM, 2 μL of sample to be detected is added, and lambda exonuclease is added to make the concentration in the system 125 U / mL. If the total volume of the system is less than 20 μL, it is supplemented with ddH2O. It is placed in a real-time fluorescence detector at 37 ℃ for detection. The detection time interval is set to 10 seconds per cycle, the cycle number is 600, and the detection time is 100 minutes. The fluorescence intensity-time trajectory is shown in (C) of FIG. 6. Figure 3

[0150] The fluorescence value-time trajectory of the reaction process is captured and recorded by the real-time fluorescence instrument, and the output is a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0151] ​When there is no double-stranded target in the sample to be tested, the fluorescence value does not change significantly; when there is a double-stranded target in the sample to be tested, the fluorescence value of the four different functional group modified probes increases significantly with time, and the fluorescence value of the carboxyl modified probe is the highest.

[0152] Example 4

[0153] Using type I double-stranded nucleic acid detection probe to detect non-small cell lung cancer related gene EGFR-L858R.

[0154] This example is based on type I double-stranded nucleic acid detection probe for detection, the specific steps are as follows:

[0155] 1、The specific detection probe used in this example is type I double-stranded nucleic acid detection probe P7, which is specific for non-small cell lung cancer related gene EGFR-L858R, and is composed of Dz1 (see Table 1) and R7 according to a molar ratio of 1:1, and the specific sequence of R7 is shown in Table 4:

[0156]

[0157] Note: COOH in Table 4 represents carboxylic acid modification.

[0158] The target gene is EGFR-L858R, one strand of the double-stranded target thereon has a length of 45 nucleotides, and the sequence is the same as the 1st-45th of R7, and the other strand has a sequence that is reverse complementary to the 1st-45th of R7.

[0159] The structure of probe P7 is shown in Figure 1 (C) of the above figure, which is composed of two DNA single strands of R7 and Dz1, and the molar ratio of R7 to Dz1 is 1:1.

[0160] The sequence of R7 is shown in SEQ ID No: 15 in the sequence listing, and has a length of 72 nucleotides, and the 1st is modified with a carboxylic acid group, and R7 is connected in the 5'-3' direction by a target binding domain (1st-45th) and a Dz binding domain (46th-72nd); wherein the target binding domain is reverse complementary to one strand of the double-stranded target of the target gene EGFR-L858R, and the Dz binding domain is reverse complementary to the Dz1.

[0161] According to Table 4, the required type I double-stranded nucleic acid detection probe P7 is synthesized.

[0162] 2、In the probe system containing the above-mentioned type I double-stranded nucleic acid detection probe P7, the sample to be tested, exonuclease (lambda exonuclease) and reporter molecule S1 are added to form a detection system, and the fluorescence intensity is recorded;

[0163] 2.1 Preparation of type I double-stranded DNA specific detection probe

[0164] Preparation of probe (20 μL): reaction buffer environment is 1 × lambda exonuclease buffer, and type I double-stranded nucleic acid detection probe P7 is added to make the concentration of each single-stranded DNA in type I double-stranded nucleic acid detection probe P7 in the system be 1 μM. The total volume of the system is equal to 20 μL, and the rest of the volume is supplemented with ddH2O.

[0165] An annealing program is used to ensure that the oligonucleotide chains constituting the probe are correctly combined. The annealing program setting program is as follows: 90°C, 2 min; 85°C, 1 min; 80°C, 1 min; 75°C, 1 min; 70°C, 1 min; 65°C, 1 min; 60°C, 1 min; 55°C, 1 min; 50°C, 1 min; 45°C, 1 min; 40°C, 1 min; 37°C, 2 min; 16°C, 1 min; 4°C, hold.

[0166] The samples to be tested used are as follows: tumor tissue samples of non-small cell lung cancer patients (from Tianjin Chest Hospital, 20 people), and normal tissue samples of healthy people's lungs (from Tianjin Chest Hospital, 10 people) are taken respectively. The tissue DNA is extracted by using a nucleic acid extraction kit (MolPure ® Blood / Cell / Tissue / Bacteria DNA fast Kit) to obtain genomic DNA as the sample to be tested from the patient and the sample to be tested from the healthy person, and a blank control is set.

[0167] 2.2 Detection

[0168] 20 μL detection system: 2 μL of the above type I double-stranded nucleic acid detection probe P7 with a concentration of 1 μM is taken to make the concentration in the system be 100 nM, 10 μL of the sample to be tested is added, lambda exonuclease is added to make the concentration in the system be 125 U / mL, and reporter molecule S1 is added to make the concentration in the system be 500 nM. If the total volume of the system is less than 20 μL, it is supplemented with ddH2O. It is placed in a real-time fluorescence detector under the condition of 37°C for detection, and the detection time interval is set to 10 seconds once, the cycle number is 900, the detection time is about 150 minutes, and the fluorescence intensity-time trajectory is obtained. When there is no target nucleic acid sequence in the detection target, the fluorescence value does not change significantly; if the fluorescence value rises significantly, it is considered that there is a target nucleic acid sequence in the detection target.

[0169] 2.3 Analysis of fluorescence intensity

[0170] The fluorescence intensity recorded in the detection is analyzed to determine whether there is a double-stranded DNA double-stranded target in the sample to be tested:

[0171] Figure 4The results show that if the double-stranded target exists in the sample to be tested, the fluorescence value increases significantly; if the double-stranded target does not exist in the sample to be tested, the fluorescence value does not increase significantly.

[0172] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In general, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A composition for detecting double-stranded nucleic acid, characterized by, The composition comprises a double-stranded nucleic acid detection probe, a lambda exonuclease and a reporter molecule; The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target exists in the sample to be detected; the double-stranded nucleic acid detection probe comprises a single-stranded DNA named recognition strand R and n single-stranded DNAs named Dz connected with the recognition strand R through base complementary pairing; the recognition strand R comprises a target binding domain and n Dz binding domains arranged in sequence from 5' to 3' direction; n Dz binding domains and the recognition strand R form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; the 5' end of the recognition strand R is modified with a functional group to realize the combination of the double-stranded nucleic acid detection probe and the lambda exonuclease; the Dz is a deoxyribozyme capable of enzymatically cutting the reporter molecule, and the 5' end of the Dz is modified to prevent the Dz from being degraded by the lambda exonuclease; The reporter molecule is a specific substrate of the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quencher group; The deoxyribozyme is selected from any one of 10-23 DNA enzyme, 8-17 DNA enzyme, 17E DNA enzyme, Mg5 DNA enzyme, 39E DNA enzyme, EtNa DNA enzyme and Ce13d DNA enzyme; the core catalytic domain sequence of the 10-23 DNA enzyme is shown in SEQ ID No: 1 of the sequence table, wherein the letter M represents A or C, the letter H represents A, C or T, N represents A, C, G or T, and D represents A, G or T; the core catalytic domain of the 8-17 DNA enzyme is any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3' and 5'-NNNNAGYNNNNCGN-3', wherein the letter N represents A, C, G or T, and the letter Y represents T or C; The n is a natural number of 1-10; The functional group is any one of a phosphate group, a carboxyl group, an amino group and a sulfhydryl group; The quencher group is BHQ1, and the fluorescent group is FAM; The composition comprises any one of P1-P7: P1, the double-stranded target is a double strand composed of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10; the double-stranded nucleic acid detection probe is formed by the combination of a single-stranded DNA with the sequence of SEQ ID No: 2 and a single-stranded DNA with the sequence of SEQ ID No: 4 through base complementary pairing, and the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 6; P2, the double-stranded target is a double strand consisting of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of a single-stranded DNA with the sequence of SEQ ID No: 3 and a single-stranded DNA with the sequence of SEQ ID No: 5; the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 7; P3, the double-stranded target is a double strand consisting of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of a single-stranded DNA with the sequence of SEQ ID No: 11 and two single-stranded DNAs with the sequence of SEQ ID No: 4; the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 6; P4, the double-stranded target is a double strand consisting of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of a single-stranded DNA with the sequence of SEQ ID No: 12 and a single-stranded DNA with the sequence of SEQ ID No: 4; the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 6; P5, the double-stranded target is a double strand consisting of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of a single-stranded DNA with the sequence of SEQ ID No: 13 and a single-stranded DNA with the sequence of SEQ ID No: 4; the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 6; P6, the double-stranded target is a double strand consisting of a single-stranded DNA with the sequence of SEQ ID No: 8 and a single-stranded DNA with the sequence of SEQ ID No: 9 or a single-stranded RNA with the sequence of SEQ ID No: 10, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of a single-stranded DNA with the sequence of SEQ ID No: 14 and a single-stranded DNA with the sequence of SEQ ID No: 4; the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No: 6; P7, the double-stranded target consisting of a single-stranded DNA with sequence of SEQ ID No: 15 1-45 and a single-stranded DNA with sequence of reverse complement of SEQ ID No: 15 1-45, the double-stranded nucleic acid detection probe is formed by base complementary pairing combination of one single-stranded DNA with sequence of SEQ ID No: 15 and one single-stranded DNA with sequence of SEQ ID No: 4, and the corresponding specific substrate is a DNA-RNA chimera with sequence of SEQ ID No:

6.

2. Use, characterized in that, The application is the use of the composition of claim 1 in the preparation of a nucleic acid detection reagent.

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